Understanding how distinct classes of motor neurons are specified and guided to appropriate muscle domains is of fundamental importance to the design of therapeutic approaches to nerve regeneration and motor neuron disease. It is well established that combinatorial codes of transcription factors dictate motor axon pathway selection in both vertebrate and invertebrate nervous systems; however, the downstream targets of these transcription factor codes that control axon guidance are poorly defined. This proposal seeks to define functional links between transcriptional regulators of motor axon guidance and specific axon guidance receptors that control path finding. The Drosophila embryonic nervous system is an attractive model in which to address these questions because of the ability to label and genetically manipulate individual and uniquely identified motor neurons. In addition, many of the key transcriptional regulators as well as the guidance cues and receptors are evolutionarily conserved; thus, findings in Drosophila are very likely to be directly relevant to higher vertebrate nervous systems. The major aims of this proposal are: 1) to determine whether the even-skipped homeobox transcription factor directly or indirectly regulates the expression of the Unc-5 axon guidance receptor to guide motor axons dorsally, 2) to determine whether the Slit receptor Robo2's influence on motor axons that project to ventral muscle targets reflects a functional link with the transcriptional regulation of ventral projection, and 3) to identify additional downstream targets of the transcriptional regulators of dorsal motor axon pathway selection using complementary genetic and molecular screening approaches. Classical genetic and biochemical techniques, including genetic interaction tests, mutant analysis, and mis-expression experiments will be used to investigate the role of Slit and Netrin receptors in contributing to the readout of the transcriptional code for motor axon guidance. To identify additional determinants of dorsal motor axon projection, we will take advantage of dose-dependent effects of mis-expressing even-skipped to 1) perform genetic screens for dominant enhancers and suppressors of this mis-expression phenotypes and 2) perform mRNA expression screens using candidate genes and cDNA microarrays for genes that are differentially regulated by even-skipped in purified populations of dorsally projecting motor neurons. PUBLIC HEALTH RELEVANCE: The proposed research has the potential to make important contributions to the understanding of developmental disorders of the nervous system and may suggest new strategies to promote regeneration after brain and spinal cord injury. In addition, a more complete understanding of how neurons develop and form specific connections will be invaluable for developing stem cell therapies for neuronal replacement to treat neurological disorders ranging from Alzheimer's disease to Amyotrophic Lateral Sclerosis. [unreadable] [unreadable]